2.1 Phospholipase A.sub.2 Proteins
Phospholipase A2s (PLA2s) are enzymes that catalyze the hydrolysis of the sn-2 fatty acyl ester bond of membrane phospholipids to yield a free fatty acid, e.g., arachidonic acid and a lysophospholipid. Both the arachidonic acid and the lysophospholipids can be further metabolized to produce inflammatory mediators. The products of arachidonic acid metabolism are eicosanoids: lipoxin, HETES, leukotrienes and prostaglandins. Leukotrienes recruit inflammatory cells and promote extravasation of these cells into injured sites and promote epidermal proliferation. Leukotrienes also stimulate the release of superoxide and proteases that damage tissue at an inflamed site. Prostaglandins increase blood flow allowing infiltration of leukocytes to inflamed sites and potentiate the pain response. The 2-acetyl derivative of 1-O-alkyl-glycero-3-phosphocholine lysophospholipid metabolism is Platelet Activating Factor (PAF). PAF causes aggregation of platelets, neutrophils, and monocytes and is also a very potent eosinophilic chemotactic factor.
There are three types of PLA2 proteins in mammals including humans. There is a cytoplasmic PLA2 having a molecular weight of about 85 KD. There is also a class of closely related low molecular weight PLA2 proteins. Low molecular weight PLA2s are characterized by a millimolar catalytic requirement for calcium and a rigid three dimensional structure maintained by multiple disulfide bridges. These proteins, which have a uniform molecular weight of 14 KD and contain an invariable 18 amino acids sequence. The low molecular weight phospholipases can be divided into two groups based on their amino acid sequence. Waite, M., 1987, 5 THE PHOSPHOLIPASES, HANDBOOK OF LIPID RESEARCH, ed. by D. J. Hanahan (Plenum Press, NY) pp 155-190. Type I PLA2 include mammalian pancreatic digestive enzymes find the well characterized snake venom enzymes. The type I enzyme has a disulfide bridge connecting cysteines at positions 11 and 77.
Type II PLA2s lack the disulfide bridge between positions 11 and 77 and have a carboxyl terminal 6 amino acid extension. Type II PLA2s share 30-40% amino acid sequence homology with each other. The purified human type II PLA2 enzyme is significantly more potent generator of inflammation that the type I PLA2 enzyme found in pancreas. Mammalian type II PLA2s are normally present in trace amounts in many different tissues and cell types, such as platelets, neutrophils, vascular smooth muscle cells, spleen, liver, and intestine as well as placenta. Type II PLA2s are secreted by certain cells of the immune system in response to stimulation. This type of PLA2 is found in soluble form at inflammatory sites, e.g., experimental casein or glycogen-induced peritoneal exudates, Forst, J. M., et al., 1986, BIOCHEMISTRY 25: 8381-8385; Chang, J., et al., 1987, BIOCHEM. PHARMACOL., 36: 2429-2436, and the synovial fluid of patients with rheumatoid arthritis, Vadas, P., et al., 1985, LIFE SCI. 36: 579-587; Seilhamer, J. J., et al., 1989, J. BIOL CHEM. 264: 5335-5338, Kramer, R. M., et al., 1989, J. BIOL. CHEM. 264: 5768-5775. Recombinantly produced type II PLA2 is arthritogenic when injected into the joint space of a rabbit. Bomalaski, J. S., et al., 1991, J. IMMUNOL. 146: 3904-3910. Disease conditions in man such as acute pancreatitis, septic shock, inflammatory arthritis and adult respiratory distress syndrome (ARDS), have been associated with elevated levels of secretory PLA2s. Vadas, P., & Pruzanski, W., 1986, LABORATORY INVESTIGATION 55:391-404.
These data have led to a generally recognized need for a clinically effective inhibitor of type II PLA2 enzyme activity. Garst, M. E., 1992, CURR. OPIN. THERA. PATENTS 2:400-408. See, e.g., Miyake, A., et al., 1992, J. OF PHARM. EXP. THERA. 263:1302-1307; Kreft, A., 1993, AGENTS ACTIONS 39:C33-C35.